Multiplexed Biomolecule Arrays Made By Polymer Pen Lithography

Abstract

Methods of patterning multiple biomolecules on a surface are disclosed. The method includes inking a polymer pen array, where tips are inked with selected inks comprising the biomolecules, and transferring the biomolecules to a surface using a polymer pen lithography technique. Methods of using the multiple patterned biomolecules on a surface are also disclosed.

1 . A method of simultaneously printing at least two different biomolecules on a substrate surface comprising
 coating a tip array with at least two inks by dipping the tip array into a corresponding inkwell array having a first plurality of wells comprising a first ink comprising a first biomolecule and a first carrier and a second plurality of wells comprising a second ink comprising a second biomolecule and a second carrier such that a first plurality of tips of the tip array are dipped into the first plurality of wells and coated with the first ink and the second plurality of tips of the tip array are dipped into the second plurality of wells and coated with the second ink,   the tips of the tip array comprising non-cantilevered tips each having a radius of curvature of less than about 1 μm and comprising a compressible elastomeric polymer;   contacting a substrate surface for a first contacting period of time and at a first contacting pressure with all or substantially all of the coated tips of the array to deposit the first ink onto the substrate surface at a set of first positions to form a first set of indicia and the second ink onto the substrate surface at a set of second positions to form a second set of indicia, the all of the indicia of the first and second sets being substantially uniform in size.   
     
     
         2 . The method of  claim 1 , further comprising at least partially filling the first plurality of wells with the first ink and at least partially filling the second plurality of wells with the second ink by jetting droplets of ink into the wells using an inkjet printer. 
     
     
         3 . The method of  claim 2 , wherein the inkjet printer is an electrohydrodynamic inkjet printer. 
     
     
         4 . The method of  claim 1 , wherein all of the indicia of the first and second sets are substantially uniform in ink density. 
     
     
         5 . The method of  claim 1 , wherein the inkwell has inter-well spacings, well dimensions, or both, which correspond to tip apex spacings, tip dimensions, or both, of the tips of the tip array, respectively. 
     
     
         6 . The method of  claim 1 , wherein at least one apex of a tip of the first plurality of tips and at least one apex of a tip of the second plurality of tips are separated by a distance of less than 200 μm. 
     
     
         7 . The method of  claim 6 , wherein an indicium of the first set and an indicium of the second set are separated on the surface by a distance of less than 100 μm. 
     
     
         8 . The method of  claim 1 , wherein the first biomolecule, the second biomolecule, or each of the first biomolecule and the second biomolecule comprises an antibody, antigen, protein, enzyme, peptide, oligonucleotide, polynucleotide, oligosaccharide, polysaccharide, or mixture thereof. 
     
     
         9 . The method of  claim 1 , comprising coating the tip array with no or substantially no contamination of the first ink to the second plurality of tips. 
     
     
         10 . The method of  claim 1 , comprising forming the first set of indicia with no or substantially no contamination of the second ink. 
     
     
         11 . The method of  claim 1 , wherein the first ink, the second ink, or each of the first ink and second ink comprises glycerol, polyethylene glycol, or a mixture thereof. 
     
     
         12 . The method of  claim 1 , wherein at least the well side of the inkwell array comprises a fluorinated surface. 
     
     
         13 . The method of  claim 12 , wherein the fluorinated surface comprises a fluorinated silane. 
     
     
         14 . The method of  claim 13 , wherein the fluorinated silane comprises 1H,1H,2H,2H-perfluorodecyltrichlorsilane. 
     
     
         15 . The method of  claim 1 , comprising forming the first set of indicia, the second set of indicia, or both with a feature size of less than 1 μm. 
     
     
         16 . The method of  claim 1 , wherein the first biomolecule, the second biomolecule, or both further comprise a label. 
     
     
         17 . The method of  claim 16 , wherein the label is a fluorescent label. 
     
     
         18 . The method of  claim 17 , wherein the fluorescent label is selected from the group consisting of a fluorescein dye, 6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid, 5(and 6)-carboxy-X-rhodamine, a rhodamine dye, a benzophenoxazine, Cyanine 2 (Cy2) dye, Cyanine 3 (Cy3) dye, Cyanine 3.5 (Cy3.5) dye, Cyanine 5 (Cy5) dye, Cyanine 5.5 (Cy5.5) dye, Cyanine 7 (Cy7) dye, Cyanine 9 (Cy9) dye, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5(6)-carboxy-tetramethyl rhodamine, and combinations thereof. 
     
     
         19 . The method of  claim 1 , wherein the first biomolecule comprises a first label, and the second biomolecule comprises a second label different from the first label. 
     
     
         20 . The method of  claim 1 , wherein each tip has a radius of curvature of less than about 0.2 μm. 
     
     
         21 . The method of  claim 1 , wherein the compressible elastomeric polymer of the tip array has a compression modulus in a range of about 10 MPa to about 300 MPa. 
     
     
         22 . The method of  claim 1 , wherein the compressible elastomeric polymer comprises polydimethylsiloxane (PMDS). 
     
     
         23 . The method of  claim 22 , wherein the PMDS comprises a trimethylsiloxy terminated vinylmethylsiloxane-dimethysiloxane copolymer, a methylhydrosiloxane-dimethylsiloxane copolymer, or a mixture thereof. 
     
     
         24 . The method of  claim 1 , wherein each tip of the tip array is identically-shaped. 
     
     
         25 . The method of  claim 24 , wherein the tip shape is pyramidal. 
     
     
         26 . The method of  claim 24 , wherein the wells are pyramidal. 
     
     
         27 . The method of  claim 1 , further comprising moving the tip array, the substrate surface, or both, with respect to each other, and repeating the contacting step for a second contacting period of time, same or different from the first contacting period of time and at a second contacting pressure, same or different from the first contacting pressure. 
     
     
         28 . The method of  claim 1 , comprising limiting lateral movement between the tip array and the substrate during the contacting step, to form indicia comprising dots. 
     
     
         29 . The method of  claim 28 , comprising controlling the contacting period of time, the contacting pressure, or both to form the dots with a diameter in a range of about 10 nm to about 500 μm. 
     
     
         30 . The method of  claim 1 , comprising simultaneously contacting each tip of the tip array with the substrate surface. 
     
     
         31 . The method of  claim 1 , wherein the tip array further comprises a third plurality of tips and the inkwell array comprises a third plurality of wells comprising a third ink comprising a third biomolecule and a third carrier, and further comprising coating the third plurality of tips during said dipping step and printing the third biomolecule on the substrate surface during said contacting step, to form a third set of indicia at a set of third positions, wherein all of the indicia of the third set are substantially uniform in size with the first set of indicia and the second set of indicia. 
     
     
         32 . The method of  claim 31 , wherein all of the indicia of the third set are substantially uniform in biomolecule density with the first set of indicia or the second set of indicia. 
     
     
         33 . The method of  claim 32 , wherein all of the indicia of the third set are substantially uniform in biomolecule density with the first set of indicia and the second set of indicia. 
     
     
         34 . The method of  claim 1 , further comprising leveling the tips of the tip array with respect to the substrate surface by
 backlighting the tip array with incident light to cause internal reflection of the incident light from the internal surfaces of the tips;   bringing the tips of the tip array and the substrate surface together along a z-axis up to a point of contact between a subset of the tips with the substrate surface, contact indicated by increased intensity of reflected light from the subset of tips in contact with the substrate surface, whereas no change in the intensity of reflected light from other tips indicates non-contacting tips; and   tilting one or both of the tip array and the substrate surface with respect to the other in response to differences in intensity of the reflected light from the internal surfaces of the tips, to achieve contact between the substrate surface and non-contacting tips, wherein said tilting is performed one or more times along x-, y-, and/or z-axes.   
     
     
         35 . The method of  claim 1 , further comprising leveling the tips of the tip array with respect to the substrate surface by
 backlighting the tip array with incident light to cause internal reflection of the incident light from the internal surfaces of the tips;   bringing the tips of the tip array and the substrate surface together along a z-axis to cause contact between the tips of the tip array and the substrate surface;   further moving one or both of the tip array and the substrate towards the other along the z-axis to compress a subset of the tips, whereby the intensity of the reflected light from the tips increases as a function of the degree of compression of the tips against the substrate surface; and   tilting one or both of the tip array and the substrate surface with respect to the other in response to differences in intensity of the reflected light from internal surfaces of the tips, to achieve substantially uniform contact between the substrate surface and tips, wherein said tilting is performed one or more times along x-, y- and/or z-axes.   
     
     
         36 . The method  claim 1 , further comprising
 forming a master comprising an array of recesses in a substrate separated by lands;   filling the recesses and covering the lands with a prepolymer mixture comprising an prepolymer and, optionally, a crosslinker;   covering the filled and coated substrate with a planar glass layer;   curing the prepolymer mixture to form a polymer structure that comprises the tip array and common substrate;   removing the cured polymer structure from the master; and   at least partially filling the recesses of the master with one or more inks for use as an inkwell array for the tip array.   
     
     
         37 . The method of  claim 1 , further comprising fabricating a mold having recesses and lands; forming a tip array with the mold; removing the formed tip array from the mold; at least partially filling the recesses of the mold with one or more inks to form an inkwell array; and then coating a tip array with said inks by dipping the tip array into the inkwell array. 
     
     
         38 . The method of  claim 36 , further comprising treating at least the surface of the master comprising said recesses and lands with a fluorinated substance. 
     
     
         39 . The method of  claim 38 , comprising carrying out the treating prior to filling the master with a prepolymer mixture. 
     
     
         40 . The method of  claim 38 , comprising carrying out the treating after filling the master with a prepolymer mixture. 
     
     
         41 . The method of  claim 38 , wherein the fluorinated substance comprises 1H,1H,2H,2H-perfluorodecyltrichlorsilane. 
     
     
         42 . An article comprising
 a substrate;   a first set of indicia on the substrate surface comprising a first biomolecule, and a second set of indicia on the substrate surface comprising a second biomolecule,   
       wherein all of the indicia of the first set and the second set are substantially uniform in size and an indicium of the first set and an indicium of the second set are separated on the surface by a distance of less than 200 μm. 
     
     
         43 . The article of  claim 42 , wherein all of the indicia of the first set and the second set are substantially uniform in density. 
     
     
         44 . The article of  claim 42 , wherein an indicium of the first set and an indicium of the second set are separated on the surface by a distance of less than 100 μm. 
     
     
         45 . The method of  claim 42 , wherein all of the indicia of the first and second sets have a feature size of less than 100 μm. 
     
     
         46 . The article of  claim 42 , wherein the first biomolecule, the second biomolecule, or each of the first biomolecule and the second biomolecule comprises an antibody, antigen, protein, enzyme, peptide, oligonucleotide, polynucleotide, oligosaccharide, polysaccharide, or mixture thereof. 
     
     
         47 . The article of  claim 42 , further comprising a third set of indicia on the substrate surface comprising a third biomolecule, wherein all of the indicia of the third and all of the indicia of the first set are substantially uniform in size. 
     
     
         48 . The article of  claim 47 , wherein all of the indicia of the third set and all of the indicia of the first set are substantially uniform in density.